Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by real-time RT-PCR assay using self-quenched fluorogenic primers

Abstract

HIV-1 viral load is a basic marker to evaluate the severity of HIV-1 related diseases and to monitor the effectiveness of treatment. A method based on real-time RT-PCR technology has been developed to quantify HIV-1 RNA using self-quenched fluorogenic primers known as LUX™ primers. They were used in this study to recognize a low variable gag region of subtype E and B consensus sequences. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with 10 fold serial dilutions of synthetic HIV-gag RNA. A broad range linear relationship (10 to 106 copies/ml) was observed between the number of PCR cycles needed to detect a fluorescent signal and the number of RNA copies. The intra- and inter-assay coefficients of variation were 0.72 to 2.54% and 3.14 to 8.83%, respectively, thus indicating good reproducibility. Thirty out of fifty HIV-infected individual plasma samples were quantified by this method and compared with the AMPLICOR® HIV-1 Monitor assay, which is widely considered the reference technique for HIV-RNA viral load measurement. The results indicate that the AMPLICOR® HIV-1 Monitor assay and real-time RT-PCR using LUX™ primers are in good agreement (mean difference in log 10 copies/ml ± 2 standard deviations = 0.21 ± 1.34).